In the detection, diagnosis and treatment of diseases, it is highly important to simply and quickly detect the presence of certain ingredients contained in blood, lymph, urine and other body fluids while determining the quantity thereof.
For example, quick and simple determination of the quantity of glucose in a body fluid such as urine or blood is a prime desideratum to the early detection, diagnosis and control of diabetes. Quick and simple determination of the quantity of the protein in a body fluid, particularly urine assumes an important role in the early detection, diagnosis and treatment of gastropathy. Accurate determination of the pH of a body fluid, particularly urine, can aid not only in detection of the presence of protein therein but also in confirmation of the possible bacteriuria which induces pyelitis, cystitis and like urosis. Further, detection of the presence of occult blood in a body fluid, particularly urine, contributes greatly to the early detection, diagnosis and treatment of nephropathy, while determination of the quantity of urobilinogen in a body fluid, particularly urine, holds an important position in the diagnosis of hepatic insufficiency.
As indicated above, it is of crucial importance to simply and quickly detect glucose, protein, occult blood and urobilinogen in a body fluid, particularly urine, or determine the pH of the body fluid. For this purpose, an indicator strip comprising an indicator reagent-impregnated filter paper affixed to a support has heretofore been used in most cases. This type of indicator strip has the advantageous features that it can be handled easily and that the results can be obtained in a short period of time.
In an indicator strip for detecting glucose in a body fluid, especially urine, the glucose entrapped therein reacts with oxygen in the air through action of a glucose-oxidizing enzyme to be finally oxidized into gluconic acid and hydrogen peroxide. The hydrogen peroxide, in turn, generates nascent oxygen through the action of peroxidase, which oxygen reacts immediately with an oxidizable indicator such as o-tolidine to cause the indicator to form color.
The indicator strip for detecting glucose in a body fluid based on the above mechanism was prepared by dissolving or dispersing in water or a water-alcohol solvent system a reagent composition comprising a glucose oxidase, peroxidase and an oxidizable indicator, impregnating a filter paper with the resultant solution, drying the filter paper, causing the filter paper to adhere to a plastic film, and cutting the laminate thus formed to a suitable size.
This method, however, is accompanied by the following problems.
(a) In the case where an impregnant solution is prepared by dissolving or dispersing in water or a water-alcohol solvent system a reagent composition comprising a glucose oxidase, peroxidase and an oxidizable indicator, the enzyme is unstable and hence is liable to be deactivated while the impregnant solution deteriorates rapidly. For this reason, multiple-step impregnation of the filter paper is required immediately after the preparation of the impregnant solution. There remains another problem that, even if the impregnation of the filter paper is promptly carried out, a part of the enzyme is deactivated and a part of the impregnant solution deteriorates.
(b) Since the impregnant solution is unstable, and also complicated process steps are required as has been set forth hereinabove, it is difficult to maintain uniform quality of the product indicator strip, particular caution and skill are required in order to secure the precision and reliability of the indicator strip, the process efficiency tends to be lowered, and the production costs are raised.
Accordingly, there have been attempts to develop indicator strips which can be obtained by a simplified process and is suitable for mass production. Japanese Patent Pub. No.25953/1969 discloses a process for the fabrication of indicator strips by dissolving enzymes in a water-alcohol solvent mixture in advance, admixing therewith an indicator, a pH buffer, a polymer binder and a water-absorptive carrier to prepare an ink composition suitable for printing or coating, applying the ink composition onto a support by printing (including coating), and drying the ink composition thus applied onto the support. In this process, however, enzymes partly dissolved in water are unstable and become rapidly deactivated, so that printing must be carried out immediately after the preparation of the ink composition while at the same time the ink composition must be dried at a low temperature to prevent deactivation of the enzymes and the residual water content of the applied reagent layer must be minimized to obtain good shelf life.
Under the circumstances, we have proposed in Japanese Patent Laid-Open Pub. No.209995/1983 a process for the fabrication of indicator strips by dispersing enzymes in a nonaqueous solvent which does not substantially dissolve the enzymes, and subsequently dissolving or dispersing an indicator, a buffer, a binder and a water-absorptive carrier to prepare an ink composition, and applying the ink composition onto a support by printing. In accordance with this process, an indicator strip exhibiting remarkable ability to measure the quantity of glucose and also having high sensitivity can be obtained, but this indicator strip, when exposed to the air for a long period of time, turns color gradually. Since the color turning reaction of the indicator strip during storage in the air may lead to an incorrect indication in the examination of body fluids, there has been an urgent demand for a solution to this problem.
As a result of our research conducted in an effort to solve the problem set forth hereinbefore, we have found that the color turning reaction of the aforementioned indicator strip for detecting glucose is attributable to the effect of peroxides and the like present in the air in trace amounts on the reagent composition, particularly the oxidizable indicator contained therein. As a result of our subsequent research, we have further found that this color reaction can be inhibited by adding a stabilizer to the reagent composition, and that stabilizer compounds having adequate antioxidant activity and specific surfactants are especially remarkable stabilizers.
On the other hand, an indicator strip for detecting protein in a body fluid, particularly urine, has been conventionally fabricated by immersing a water-absorptive carrier in a solution comprising an indicator which indicates a protein error and a buffer, drying the carrier, and adhering the carrier to a support. This process, however, is still accompanied by the problem that the process steps are complicated. Thus, an indicator strip for detecting protein which can be obtained by a simplified process has been proposed. For example, Japanese Utility Model Laid-Open Pub. No.79767/1982 discloses an indicator strip for detecting protein fabricated by applying on a support a reagent composition comprising a pH indicator, a buffer, a binder and a water-absorptive powder by means of pattern printing. However, this indicator strip has the drawbacks that, due to low permeability and poor water retention, protein in a body fluid does not contact with the portion of the indicator strip where the reagent has been applied and hence the sensitivity of the strip is low and it takes a long time to form color after the strip is immersed in a body fluid. Another drawback of this indicator strip is that a fading phenomenon is conspicuously observable when the reagent portion of the strip which has been immersed in a body fluid to be tested is dried.
As a result of our intensive investigation pursued to solve the above described problems, we have found that, by adding an ion exchange resin having an affinity for protein and also exhibiting adsorptive action to a reagent composition for detecting protein, all of the problems incident to the conventional indicator strips can be solved, thus providing a highly sensitive indicator strip for detecting protein capable of forming a distinct color in a short period of time, which color undergoes almost no fading after drying.
An indicator strip for determining the pH of a body fluid, particularly urine, has hitherto been produced by impregnating a water-absorptive support with an aqueous solution containing a plurality of pH indicators and thereafter drying. This indicator strip has the disadvantage of high production costs due to complicated process steps and hence difficult process control. In order to eliminate this disadvantage, an indicator strip for the pH determination which can be obtained by a simplified process has been proposed. Japanese Patent Pub. No. 25953/1969, for instance, describes an indicator strip comprising a plurality of pH indicators and an adsorptive powder both adhered to a support. This indicator strip is again accompanied by the problems set forth hereinbelow.
(a) When the indicator strip is immersed in a body fluid to be tested and then dried, a color which has once been formed fades conspicuously.
(b) When the indicator strip is immersed in a body fluid to be tested, the indicators may sometimes be eluted into the body fluid, so that a distinct color may not be formed in some cases.
(c) When the indicator strip is immersed in a body fluid to be tested, it takes a long time to form color.
We have further conducted a research to solve the above problems and found that all of these problems can be solved by applying an ink composition for the pH determination comprising a pH indicator, a quaternary ammonium salt or an amine salt, a binder, a water-absorptive powder and a solvent onto a support.
As mentioned previously, it is important to determine the quantity of occult blood or urobilinogen in a body fluid, particularly urine, in checking the renal or hepatic function. An indicator strip for detecting occult blood or urobilinogen has been conventionally formed by coating a support with a reagent composition for use in the detection comprising an indicator for detecting either one of the body fluid ingredients, a water-absorptive carrier and water or a water-alcohol solvent mixture. In the case where water or a water-alcohol solvent mixture is incorporated in a reagent composition, however, the composition thus applied onto the support must be heated to a considerably high temperature in the subsequent drying step. For this purpose, a drying apparatus is required, and, on the other hand, there is a possibility of the indicator for detecting occult blood or urobilinogen deteriorating or becoming denatured.
As a result of our extensive research, we have found that the foregoing problem can be completely solved by using, in the case of occult blood detection, a reagent composition comprising an oxidizable indicator, an organic peroxide, an occult blood detection composition binder and a nonaqueous solvent for the occult blood detection composition; and in the case of urobilinogen detection, a reagent composition comprising a color precursor which forms color by reaction with urobilinogen, a strongly acidic buffer, a urobilinogen detection composition binder and a nonaqueous solvent for the urobilinogen detection composition.
The present invention is also directed to a test device for body fluids comprising a support having printed or coated thereon at least one detection region, each detection region comprising an ink composition selection from the group of ink compositions consisting of:
(a) a glucose detection reagent in accordance with the present invention;
(b) a protein detection reagent in accordance with the present invention;
(c) a pH determination reagent in accordance with the present invention;
(d) an occult blood detection reagent in accordance with the present invention; and
(e) an urobilinogen detection reagent in accordance with the present invention.
Finally, the present invention is also directed to a test device comprising a support having printed or coated thereon at least one detection region, wherein water-retentive pattern portions are formed around each of said detection regions.